The pituitary gland is known as the master endocrine gland and controls multiple hormonal responses including those regulating reproduction, homeostasis, and responses to stress. The hormone-secreting lobe of the pituitary gland, or adenohypophysis, forms at the anterior end of the developing embryo through inductive interactions between neurally and epidermally derived tissues. Hedgehog (Hh) signaling molecules help mediate these inductive events, a role that has been conserved across vertebrate species from fish to mammals. Human mutations in Hh signaling lead to a variety of syndromes that affect pituitary development, including Holoprosencephaly and Pallister-Hall syndrome. In fact, human congenital pituitary defects are quite common and range from the loss of all endocrine function (panhypopituitarism) to the loss of single hormone function. The loss of GH is the most common single endocrine deficiency in humans, occurring in 1 in 4000 embryos. We previously showed that this range of pituitary defects also occurs in zebrafish Hh pathway mutants, providing a unique resource for the study of pituitary development. Among these, the uncharacterized uml mutation eliminates some cell types (e.g. GH) and uniquely affects cell fate decisions in the pituitary. We also showed that Gli mediated Hh signaling is needed for pituitary induction and endocrine cell differentiation. Here we propose to continue our use of the zebrafish as a model system to investigate the molecular and cellular mechanisms of Hh regulated cell differentiation in the vertebrate pituitary gland. We will first test whether Hh acts as a morphogen or mitogen in pituitary development and determine which Gli transcription factors mediate the pituitary Hh response. Using newly developed techniques to temporally and cell-autonomously disrupt Hh signaling, we will then test the direct requirement for Hh signaling in pituitary precursor cells and endocrine cell lineages and determine when direct Hh signaling is needed for these cell differentiation events. Finally, we will determine the molecular basis of the zebrafish umleitung (uml) mutation as part of a genetic investigation of Hh involvement in endocrine cell lineage determination. This work will provide fundamental knowledge about the role of Hh signaling in guiding cell specification in the vertebrate pituitary. Our research plan takes advantage of zebrafish to combine genetic, cellular, and molecular analyses at a level not possible in other vertebrates. Our new genetic tools will be useful to researchers investigating Hh in any embryonic tissue. The characterization of a potentially novel regulator of Hh signaling (the uml locus) is also likely to provide new insights into the regulation of Hh signaling throughout the developing embryo. Because the Hh signaling pathway has been highly conserved through evolution, this work will apply directly to our understanding of Hh signaling in higher vertebrates and will impact on our ability to direct stem cell differentiation for therapeutic purposes. Ultimately, this zebrafish research promises to contribute to our understanding of human birth defects affecting the pituitary and may shed light on tumorigenesis caused by mis-regulation of Hh signaling postnatally. [unreadable] [unreadable]